ABSTRACT Mycobacterium tuberculosis (Mtb) remains the leading cause of death due to infectious disease and is estimated to infect one-third of the world. Tuberculosis (TB) is a chronic, progressive disease, often with a long period of latency following initial infection. In most cases, the host immune system is capable of restraining but not eliminating Mtb, leading to lifelong infection. Mtb infection can be treated with antibiotics. However, unlike most other bacterial infections, effective TB treatment requires a combination of four drugs taken for a minimum of six months. This lengthy treatment regimen is a critical barrier to effective TB control, and is thought to be necessitated by the presence of antibiotic-tolerant bacilli that arise during infection. These combined abilities: 1) to persist in the face of strong, cell-mediated immunity and sustain chronic infection; and 2) tolerate prolonged exposure to lethal concentrations of antibiotic, are central to the continued global success of this pathogen. Due to the difficulties associated with traditional Mtb genetics, the biological mechanisms underpinning these phenomena remain largely unexplored. To close this knowledge gap, I recently developed a CRISPR interference (CRISPRi) platform to accelerate the genetic interrogation of Mtb pathogenesis. Here, I propose to leverage this transformative new tool to define the genetic basis for persistent Mtb infection. Specifically, I will perform a kinetic-genetic screen in a murine model of TB to identify the bacterial determinants that enable chronic Mtb infection. Subsequent functional characterization of these genes will define their mechanisms of action, thereby revealing the molecular architecture that supports chronic TB. In addition, it is well documented that the stress of host infection can induce antibiotic tolerance in Mtb. Here, I will test the hypothesis that the massively expanded and enigmatic gene family, the toxin-antitoxins, function through a divergent stringent response to mediate antibiotic tolerance in Mtb. I anticipate that these collective findings will remove important roadblocks to progress in the field by vertically advancing our scientific knowledge and technical capability. Furthermore, this work will serve as an intellectual foundation for the development of novel TB therapies capable of shortening the course of treatment and improving control of this pandemic.